DNA Damage and Repair
Cells are constantly being assaulted by a wide variety of toxic agents in the environment. These agents damage cellular DNA and produce products which can result in cellular lethality, mutagenesis or carcinogenesis. An important class of damaging agents are highly reactive free radicals. Free radicals are produced by ionizing radiation, a variety of chemicals and most importantly, by normal cellular oxidative metabolism. Thus, free radical-induced damages are the most common cellular DNA lesions. In fact, it has been estimated that each day a cell receives on average about 10,000 oxidative hits. Since many of these damages have potential deleterious biological consequences, it is important to delineate how cells process these lesions. Since free radical-induced DNA damages are common lesions, cells have evolved efficient repair mechanisms to deal with them. The lesions are recognized by a battery of repair glycosylases and endonucleases which catalyze the first steps in the base excision repair pathway. Recognition and removal of the damage are followed by repair polymerization and ligation.
The enzymes involved in base excision repair are ubiquitous with homologs being found from bacteria to humans. If a lesion is not repaired prior to its encounter with the replication fork, the lesion is now capable of blocking replication, potentially causing cell death, or directing misinsertion of an incorrect base, leading to mutation and/or carcinogenesis. Since free radicals produce hundreds of different products in DNA, it is important to be able to assess which product is potentially deleterious, that is, which ones are potentially lethal and which ones are potentially mutagenic. Because free radical-inducing agents produce so many lesions in DNA all at the same time, it is difficult if not impossible to pinpoint cause/effect relationships of individual lesions. Our approach has been to select model representative products and introduce these either chemically or by using recombinant DNA technologies into substrate and biologically active DNA molecules and assessing their biological consequences. Once a lesion is either randomly or site specifically introduced into a DNA molecule, we can then ask if there are cellular enzymes present that recognize the lesion and remove it from DNA. Using this as well as in silico approaches, we have purified bacterial, viral, fungal and human enzymes that specifically recognize and remove oxidative DNA damages. Using mutants defective in these enzymes, in combination with biologically active DNA containing specific damages, we have defined cellular repair pathways. With Dr. Jeffrey Bond, we have used bioinformatics to look at the evolution of the oxidative DNA glycosylases that recognize DNA base damages and have purified and characterized novel mammalian DNA glycosylases. In collaboration with both Drs. Bond and Doublie, we have used biochemical and crystallographic approaches to determine how these proteins interact with their substrates.
In order to assess whether an unrepaired oxidative DNA lesion has potential biological consequences, we have introduced unique lesions into single-stranded template DNA molecules and asked whether these are blocks to DNA polymerases in vitro. If a lesion is a block to DNA polymerase, it is a potentially cytotoxic lesion. We have examined a number of oxidative lesions having a variety of structural characteristics and in every case, when a lesion was a block to DNA polymerase in vitro, it was also lethal when present in biologically active single-stranded transfecting DNA. We have used a similar approach to study the potential mutagenicity of individual oxidative DNA lesions by asking which base polymerases insert opposite a lesion in vitro, and then correlating these results with the mutagenic spectrum produced by each lesion in vivo. Thus far, the in vitro and in vivo results have been in good agreement. Very interestingly, we have been able to relate the ability of DNA polymerases to bypass individual lesions, as well as misinsertion direction by the particular lesion, to its mutagenic consequences in the cell. We have also found that the sequence context within which the lesion finds itself, is extremely important in misinsertion direction as well as lesion bypass. The interactions between DNA polymerases and DNA lesions at the atomic level with two replication blocking damages, an abasic site and thymine glycol, have been elucidated by crystal structures obtained by our collaborator, Dr. Doublie.
Our current efforts are focused on visualizing in real time the entire Base Excision Repair pathway at the single molecule level. In collaboration with Dr. David Warshaw, and using Total Internal Reflectance Microscopy to visualize Quantum dot-labeled DNA glycosylases scanning DNA tightropes, we have already shown that the glycosylases insert a wedge residue into the DNA duplex to search for and locate damaged bases. We are poised to examine the interactions among all the enzymes that cooperate in base excision repair in humans.
Nelson SR, Dunn AR, Kathe SD, Warshaw DM, Wallace SS. Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases Proc Natl Acad Sci U S A. 2014 May 20;111(20):E2091-9
Strniste GF, Wallace SS. Endonucleolytic incision of x-irradiated deoxyribonucleic acid by extracts of Escherichia coli Proc Natl Acad Sci U S A. 1975 Jun;72(6):1997-2001
Ide H, Kow YW, Wallace SS. Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro Nucleic Acids Res. 1985 Nov 25;13(22):8035-52.
Doublie S, Bandaru V, Bond JP, and Wallace, SS. The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity. Proc Natl Acad Sci U S A. 2004 101(28):10284-10289.
Liu M, Bandaru V, Bond JP, Jaruga P, Zhao X, Christov PP, Burrows CJ, Rizzo CJ, Dizdaroglu M, Wallace SS. The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4925-4930.
All Wallace publications